Despite Excellent Test Characteristics of the cobas 4800 CT/NG Assay, Detection of Oropharyngeal Chlamydia trachomatis and Neisseria gonorrhoeae Remains Challenging.

Oropharyngeal Chlamydia trachomatis (CT) infections and, especially, Neisseria gonorrhoeae (NG) infections are common, but few commercial nucleic acid amplification tests (NAATs) specify extragenital samples for intended use. The test characteristics of the cobas 4800 CT/NG assay were evaluated for oropharyngeal swabs. The technical validation included analysis of the specificity, sensitivity, dynamic range, linearity, efficiency, and precision. The probability of detection curve combined with historical data enabled the estimation of potentially missed diagnoses. A clinical evaluation was performed on a subset of 2,798 clinical samples available from routine diagnostics. Results of the cobas 4800 were compared with those from in-house C. trachomatis/N. gonorrhoeae PCR assays. Discrepant samples were tested with resolver assays, and these results were considered decisive. No cross-reactivity was seen in the analytical specificity analysis. High linearity (R2 ≥ 0.983), efficiency (89% to 99%), and precision (cycle threshold [CT ] value of 0.1 to 0.9) were seen for both C. trachomatis and N. gonorrhoeae The limit of detection in oropharyngeal samples was 3.2 × 102 inclusion-forming units (IFU)/ml for C. trachomatis and 6.7 × 102 CFU/ml for N. gonorrhoeae Estimates on potentially missed diagnoses were up to 7.2% for C. trachomatis and up to 24.7% for N. gonorrhoeae Clinical sensitivity and specificity were evaluated with 25 C. trachomatis-positive, 86 N. gonorrhoeae-positive, and 264 negative samples, resulting in 100% and 99.6% for C. trachomatis and 100% and 96.7% for N. gonorrhoeae, respectively. The findings in this study demonstrate the utility of the cobas 4800 CT/NG assay for oropharyngeal samples. Despite its being a highly accurate test, the range of reported CT values, especially for N. gonorrhoeae, suggests relatively low oropharyngeal loads. Hence, consistent detection over the full range of oropharyngeal loads could be impaired.

A Single Negative Result for van Quantitative PCR on Enrichment Broth Can Replace Five Rectal Swab Cultures in Screening for Vancomycin-Resistant Enterococci.

The increased incidence of infections by vancomycin-resistant Enterococcus (VRE) causes an accumulation of patients who are either colonized with VRE or flagged as potentially colonized with VRE. Since such patients require precautionary isolation upon admission to a hospital, rapid methods to establish VRE colonization status would improve patient care and optimize hospital operation. We evaluated van quantitative PCR (qPCR) on one enrichment broth as a VRE-screening approach. We obtained 255 sets of five rectal specimens from 243 patients. The specimens were cultured using an amoxicillin-containing enrichment broth. Subsequently, a chromogenic agar was incubated and suspect colonies were inoculated on a blood agar plate and characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), followed by a vancomycin Etest in cases in which Enterococcus spp. were detected. The culturing results were compared with the outcome of van qPCR on all enrichment broths of the first rectal swab. The van qPCR was positive for 43% of the sample sets (vanA, n = 5; vanB, n = 101; vanA and vanB, n = 3). Based on culture data, 20 (7.8%) of the sets were VRE positive in at least one of five samples. The negative predictive value of van qPCR on the first enrichment broth was 99.3%. With a cutoff quantification cycle (Cq ) value of >35 to discriminate negative and positive samples, 87% of the negative patients can be identified within a day after obtaining the sample, compared to 7 days in the culturing approach. VRE screening using qPCR on one enrichment broth can quickly identify non-VRE-colonized patients and therefore decrease costs and limit unnecessary isolation restrictions.

Further evidence of the effects of global warming on lichens, particularly those with Trentepohlia phycobionts.

Increasing evidence suggests that lichens are responding to climate change in Western Europe. More epiphytic species appear to be increasing, rather than declining, as a result of global warming. Many terricolous species, in contrast, are declining. Changes to epiphytic floras are markedly more rapid in formerly heavily polluted, generally built-up or open rural areas, as compared to forested regions. Both the distribution (southern) and ecology (warmth-loving) of the newly established or increasing species seem to be determined by global warming. Epiphytic temperate to boreo-montane species appear to be relatively unaffected. Vacant niches caused by other environmental changes are showing the most pronounced effects of global warming. Species most rapidly increasing in forests, although taxonomically unrelated, all contain Trentepohlia as phycobiont in addition to having a southern distribution. This suggests that in this habitat, Trentepohlia algae, rather than the different lichen symbioses, are affected by global warming.

Evaluation of conventional and real-time PCR assays using two targets for confirmation of results of the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae test for detection of Neisseria gonorrhoeae in clinical samples.

Two conventional PCR-enzyme immunoassays (PCR-EIAs) and two real-time PCR assays (LightCycler system; Roche Diagnostics) were evaluated as confirmation assays with cppB and 16S rRNA genes as targets. Of 765 male and female genitourinary and nasopharyngeal specimens positive for Neisseria gonorrhoeae in the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR test (Roche Diagnostics), 229 (30%) were confirmed positive; 13 of these (5.7%) were lacking the cppB gene. Of the 534 samples (70%) that could not be confirmed, 81 (15%) showed a positive crossing point. However, melting curve analysis revealed an aberrant melting temperature in the LightCycler 16S rRNA assay; therefore, these samples were considered non-N. gonorrhoeae Neisseria species. Both of the 16S rRNA assays performed well, with positive predictive values of 99.1% and 100% for the PCR-EIAs and the real-time assays, respectively, and a negative predictive value of 99.8% for both. The cppB assays were compromised by the absence of the cppB gene in 5.7% of the N. gonorrhoeae-positive samples, resulting in negative predictive values of 96.8% and 97.6% for the PCR-EIAs and the real-time assays, respectively. Therefore, the 16S rRNA gene is preferable to the cppB gene as a target for confirmation assays. The melting curve analysis of the real-time assays provides useful additional information.